PROJECT SUMMARY Many cancers are currently treated by cytotoxic chemotherapies that exploit those cancers' dependence on enhanced nucleotide biosynthesis. However, the cytotoxic properties which make these compounds so efficacious in killing cancer cells also wreak havoc on normal proliferating cells and tissues. In order to understand how to exploit this vulnerability more effectively and more safely, we must focus our efforts on targets that are specifically required by cancer cell, but not normal cell, proliferation and survival. My discoveries have identified one such target ? the enzyme phosphoribosyl pyrophosphate synthetase 2 (PRPS2). PRPS2, and its homolog PRPS1, generate a critical precursor necessary for producing all nucleotides and function as a `molecular throttle' capable of increasing or decreasing the rate at which these genetic building blocks are made. This proposal seeks to unravel the molecular basis for this selectivity through use of metabolic flux analysis, elegant structure/function studies, and bioorthogonal chemistry and molecular biology approaches. Our studies will open up new avenues for understanding the metabolic vulnerabilities of cancer cells and may lead to intelligently-designed rational therapeutic strategies of the future. We will conduct our studies using models of MYC-driven lymphoma and myeloma, using both genetically- engineered mouse models and human cancer cell lines. Importantly, MYC has been characterized as the transcriptional engine of cancer and its ability to stimulate nucleotide and nucleic acid production are signature features of its pro-growth anabolic program necessary to drive malignancies in the B cell lineage. Using our genetic approaches that block PRPS2 function in MYC overexpressing cells, we can leverage this dependency to decipher the mechanistic basis for the deregulation of nucleotide metabolism in MYC-overexpressing cancer cells and uncover novel connections between critical nodes in the nucleotide metabolism network. For example, our proposed studies will elucidate the economics of nucleotide metabolism by determining how disrupting nucleotide supply affects the machineries it fuels, and vice-versa. Collectively, the proposed studies will be transformative in our understanding of the roles of these key molecules in the normal and cancer setting, and provide a new conceptual paradigm which can be the foundation for the development of the next generation of safer, more effective precision-based therapies and approaches.